In the past decade, fluorescence bioimaging in the second near-infrared (NIR-II, 1000−1700 nm) window has shown great potential in both fundamental research and preclinical diagnosis and prognosis, owing to high resolution at deep penetration and decreasing light absorption and scattering. Compared to inorganic contrast agents, NIR-II organic dyes are more promising for biological application due to low molecular mass, good biocompatibility, and flexible molecular design. In this review, current chemical structures and applications of NIR-II organic dyes (polymethine dyes and donor−acceptor−donor dyes) are discussed. In addition, rational design strategies for NIR-II organic dyes, including conjugated chain length, chain length alternations, charge transfer between terminal groups, rigid plane chemical structure, and other factors, are summarized. This review may provide guidance for further development of novel NIR-II organic dyes for bioimaging and biosensing.